Increased nucleic-acid guided cell editing in yeast

ABSTRACT

The present disclosure provides methods to increase the percentage of edited yeast cells in a cell population using nucleic-acid guided editing, and automated multi-module instruments for performing these methods.

RELATED CASES

This application claims priority to U.S. Ser. No. 62/866,041 filed 25Jun. 2019.

FIELD OF THE INVENTION

The present disclosure relates to methods and compositions to increasethe percentage of edited yeast cells in a cell population usingnucleic-acid guided editing, and automated multi-module instruments forperforming these methods and using these compositions.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences; hence gene function. The nucleasesinclude nucleic acid-guided nucleases, which enable researchers togenerate permanent edits in live cells. Of course, it is desirable toattain the highest editing rates possible in a cell population; however,in many instances the percentage of edited cells resulting from nucleicacid-guided nuclease editing can be in the single digits.

There is thus a need in the art of nucleic acid-guided nuclease editingfor improved methods, compositions, modules and instruments forincreasing the efficiency of editing. The present disclosure addressesthis need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure relates to methods, compositions, modules andautomated multi-module cell processing instruments that increase theefficiency nucleic-acid guided editing in a yeast cell population.

Thus, in some embodiments there is provided an editing vector forperforming nucleic acid-guided nuclease editing in yeast comprising: ayeast 2-μ backbone, a 2μ origin of replication; a standard constitutiveor inducible (e.g., non-minimal or non-core) promoter drivingtranscription of a gRNA sequence and donor DNA (HA) sequence followed bya terminator element 3′ to the gRNA and donor DNA sequences; a standardconstitutive (e.g., non-minimal or non-core) promoter drivingtranscription of a coding sequence for a degron-survival marker fusiongene followed by a terminator element 3′ to the degron-survival markerfusion gene; a standard constitutive or inducible (e.g., non-minimal ornon-core) promoter driving transcription of a nuclease or nucleasefusion coding sequence with a terminator element 5′ to the nucleasecoding sequence; and an origin of replication for propagation of theediting vector in bacteria.

In some aspects of this embodiment, the degron is an ubiquitin-dependentdegron and the degron is ubiquitin. In some aspects, the survival markeris selected from the group of hygromycin, blasticidin, nourseothricin orkanamycin.

Additionally there is provided in another embodiment an editing vectorfor performing nucleic acid-guided nuclease editing in yeast comprising:a yeast 2-μ backbone, a 2μ origin of replication; a standardconstitutive or inducible (e.g., non-minimal or non-core) promoterdriving transcription of a gRNA sequence and donor DNA (HA) sequencewith followed by a terminator element 3′ to the gRNA and donor DNAsequences; a minimal promoter driving transcription of a coding sequencefor a survival marker gene followed by a terminator element 3′ to thesurvival marker gene; a standard constitutive or inducible (e.g.,non-minimal or non-core) promoter driving transcription of a nucleasecoding sequence with a terminator element 5′ to the nuclease or nucleasefusion coding sequence; and an origin of replication for propagation ofthe editing vector in bacteria.

In some aspects, the minimal promoter is the URA3-d promoter, and insome aspects, the survival marker is selected from the group ofhygromycin, blasticidin, nourseothricin or kanamycin. In other aspects,the minimal promoter is the pHIS3 promoter, the pTRP1 promoter, thepLEU2 promoter, the pURA3 promoter, the pTEF1 promoter, or the pHXT7promoter. In other aspects, the promoter is a weak constitutive promotersuch as the pSSA1 promoter, the pPDA1 promoter, the pCYC1 promoter, thepTPS1 promoter, or the pSSB1 promoter.

In yet another embodiment there is provided a method for performingediting in yeast comprising: providing a population of yeast cells;transforming the population of yeast cells with a population of editingvectors, wherein each editing vector comprises: a yeast 2-μ backbone, a2μ origin of replication; a standard constitutive or inducible (e.g.,non-minimal or non-core) promoter driving transcription of a gRNAsequence and donor DNA (HA) sequence with followed by a terminatorelement 3′ to the gRNA and donor DNA sequences; a standard constitutive(e.g., non-minimal or non-core) promoter driving transcription of acoding sequence for a degron-survival marker fusion gene followed by aterminator element 3′ to the degron-survival marker fusion gene; astandard constitutive or inducible (e.g., non-minimal or non-core)promoter driving transcription of a nuclease or nuclease fusion codingsequence with a terminator element 5′ to the nuclease coding sequence;and an origin of replication for propagation of the editing vector inbacteria; growing the transformed yeast cells in selective medium toselect for cells expressing a degron-survival marker fusion protein;providing conditions to allow the transformed yeast cells to editnucleic acid sequences in the yeast cells; and growing the edited yeastcells.

In some aspects, the degron portion of the degron-survival marker fusiongene is selected from a Ura3-d degon, Ubi-R degron, Ubi-M degron, Ubi-Qdegron, Ubi-E degron, ZF1 degron, C-terminal phosphodegron; Ts-degron;lt-degron; auxin inducible degron; DD-degron, LID-degron; PSD degron,B-LID degron, and a TIPI degron.

Also provided is a method for performing editing in yeast comprising:providing a population of yeast cells; transforming the population ofyeast cells with a population of editing vectors, wherein each editingvector comprises: a yeast 2-μ backbone, a 2μ origin of replication; astandard constitutive or inducible (e.g., non-minimal or non-core)promoter driving transcription of a gRNA sequence and donor DNA (HA)sequence with followed by a terminator element 3′ to the gRNA and donorDNA sequences; a minimal promoter driving transcription of a codingsequence for a survival marker gene followed by a terminator element 3′to the survival marker gene; a standard constitutive or inducible (e.g.,non-minimal or non-core) promoter driving transcription of a nuclease ornuclease fusion coding sequence with a terminator element 5′ to thenuclease coding sequence; and an origin of replication for propagationof the editing vector in bacteria; growing the transformed yeast cellsin selective medium to select for cells expressing a survival markerprotein; providing conditions to allow the transformed yeast cells toedit nucleic acid sequences in the yeast cells; and growing the editedyeast cells.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is a representation of (i) a standard constitutive promoterdriving transcription of an antibiotic resistance gene and (ii) astandard constitutive promoter driving transcription of an antibioticresistance gene fused at its N-terminus with a ubiquitin peptide. FIG.1B is a representation of (i) a standard constitutive promoter drivingtranscription of an antibiotic resistance gene and (ii) a minimalconstitutive promoter driving transcription of an antibiotic resistancegene. FIG. 1C is an exemplary vector map of a yeast 2-μ plasmidconfigured for nucleic acid-guided nuclease editing of a yeast genome,where an antibiotic resistance gene is fused at its 5′ end to a degroncoding sequence. FIG. 1D is an exemplary vector map of a yeast 2-μplasmid configured for nucleic acid-guided nuclease editing of a yeastgenome, where the transcription of an antibiotic resistance gene isdriven by a minimal constitutive promoter.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing.

FIG. 3A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein and in relation to FIGS. 3B-3D.FIG. 3B illustrates a perspective view of one embodiment of a rotatinggrowth vial in a cell growth module housing. FIG. 3C depicts a cut-awayview of the cell growth module from FIG. 3B. FIG. 3D illustrates thecell growth module of FIG. 3B coupled to LED, detector, and temperatureregulating components.

FIG. 4A depicts retentate (top) and permeate (middle) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom). FIG. 4B depicts twoside perspective views of a reservoir assembly of a tangential flowfiltration module. FIGS. 4C-4E depict an exemplary top, with fluidic andpneumatic ports and gasket suitable for the reservoir assemblies shownin FIG. 4B.

FIGS. 5A and 5B depict the structure and components of an embodiment ofa reagent cartridge. FIG. 5C is a top perspective view of one embodimentof an exemplary flow-through electroporation device that may be part ofa reagent cartridge. FIG. 5D depicts a bottom perspective view of oneembodiment of an exemplary flow-through electroporation device that maybe part of a reagent cartridge. FIGS. 5E-5G depict a top perspectiveview, a top view of a cross section, and a side perspective view of across section of an FTEP device useful in a multi-module automated cellprocessing instrument such as that shown in FIGS. 2A-2C.

FIG. 6A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells in a solid wall device. FIG. 6B is aphotograph of a solid wall device with a permeable bottom on agar, onwhich yeast cells have been singulated and grown into clonal colonies.FIG. 6C presents photographs of yeast colony growth at various timepoints. FIGS. 6D-6F depict an embodiment of a solid wall isolationincubation and normalization (SWIIN) module. FIG. 6G depicts theembodiment of the SWIIN module in FIGS. 6D-6F further comprising aheater and a heated cover.

FIG. 7 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module for recursive yeastcell editing.

FIG. 8 is a simplified process diagram of an alternative embodiment ofan exemplary automated multi-module cell processing instrument usefulfor recursive yeast cell editing.

FIG. 9 is a graph demonstrating real-time monitoring of growth of S.cerevisiae str. s288c cell culture OD₆₀₀ employing the cell growthdevice as described in relation to FIGS. 3A-3D where a 2-paddle rotatinggrowth vial was used.

FIG. 10 is a graph plotting filtrate conductivity against filterprocessing time for a yeast culture processed in the cell concentrationdevice/module described in relation to FIGS. 4A-4E.

FIG. 11 is a bar graph showing the results of electroporation of S.cerevisiae str. s288c using an FTEP device as described in relation toFIGS. 5C-5G and a comparator electroporation method.

FIGS. 12A-12C comprise three graphs comparing the editing clonalityobtained using a yeast editing vector with an unmodified hygromycinresistance gene, a degron-fused hygromycin resistance gene, and ahygromycin resistance gene under the control of a minimal promoter.

FIGS. 13A-13C comprise three graphs comparing the editing clonalityobtained using a yeast editing vector with an unmodified G418 resistancegene, a degron-fused G418 resistance gene, and G418 resistance geneunder the control of a minimal promoter.

FIG. 14 shows plasmid copy number and edit rate of various plasmidconstructs and their edit rates.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, Calif. (1992), all ofwhich are herein incorporated in their entirety by reference for allpurposes. Nucleic acid-guided nuclease techniques can be found in, e.g.,Genome Editing and Engineering from TALENs and CRISPRs to MolecularSurgery, Appasani and Church (2018); and CRISPR: Methods and Protocols,Lindgren and Charpentier (2015); both of which are herein incorporatedin their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus (e.g., a target genomicDNA sequence or cellular target sequence) by homologous recombinationusing nucleic acid-guided nucleases. For homology-directed repair, thedonor DNA must have sufficient homology to the regions flanking the “cutsite” or site to be edited in the genomic target sequence. The length ofthe homology arm(s) will depend on, e.g., the type and size of themodification being made. In many instances and preferably, the donor DNAwill have two regions of sequence homology (e.g., two homology arms) tothe genomic target locus. Preferably, an “insert” region or “DNAsequence modification” region—the nucleic acid modification that onedesires to be introduced into a genome target locus in a cell—will belocated between two regions of homology. The DNA sequence modificationmay change one or more bases of the target genomic DNA sequence at onespecific site or multiple specific sites. A change may include changing1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300,400, or 500 or more base pairs of the genomic target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or morebase pairs of the genomic target sequence.

The terms “editing cassette”, “CREATE cassette” or “CREATE editingcassette” refers to a nucleic acid molecule comprising a coding sequencefor transcription of a guide nucleic acid or gRNA covalently linked to acoding sequence for transcription of a donor DNA or homology arm.

As used herein, “enrichment” refers to enriching for edited cells bysingulation, inducing editing, and growth of singulated cells intoterminal-sized colonies (e.g., saturation or normalization of colonygrowth).

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, nourseothricin N-acetyl transferase, chloramphenicol,erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,rifampicin, puromycin, hygromycin, blasticidin, and G418 may beemployed. In other embodiments, selectable markers include, but are notlimited to sugars such as rhamnose. human nerve growth factor receptor(detected with a MAb, such as described in U.S. Pat. No. 6,365,373);truncated human growth factor receptor (detected with MAb); mutant humandihydrofolate reductase (DHFR; fluorescent MTX substrate available);secreted alkaline phosphatase (SEAP; fluorescent substrate available);human thymidylate synthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); herpes simplex virus thymidine kinase (enablesnegative selection in yeast by 5-Fluoro-2′-deoxyuridine); humanglutathione S-transferase alpha (GSTA1; conjugates glutathione to thestem cell selective alkylator busulfan; chemoprotective selectablemarker in CD34+cells); CD24 cell surface antigen in hematopoietic stemcells; human CAD gene to confer resistance toN-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2a; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers

As used herein the term “survival marker” refers to a gene introducedinto a cell which confers to that cell the ability to survive growth ina selective medium.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., an engineered peptide antigen and a bindingtarget, with a binding affinity represented by a dissociation constantof about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about10⁻¹¹M, about 10⁻¹²M, about 10⁻¹³M, about 10⁻¹⁴M or about 10⁻¹⁵ M.

The terms “target genomic DNA sequence”, “cellular target sequence”,“target sequence”, or “genomic target locus” refer to any locus in vitroor in vivo, or in a nucleic acid (e.g., genome or episome) of a cell orpopulation of cells, in which a change of at least one nucleotide isdesired using a nucleic acid-guided nuclease editing system. The targetsequence can be a genomic locus or extrachromosomal locus.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. Theengine/editing vector for yeast as described herein comprises a codingsequence for a nuclease or nuclease fusion to be used in the nucleicacid-guided nuclease systems; a donor nucleic acid, optionally includingan alteration to the cellular target sequence that prevents nucleasebinding at a PAM or spacer in the cellular target sequence after editinghas taken place; a coding sequence for a gRNA where the gRNA iscompatible with the nuclease or nuclease fusion; and a coding sequencefor a survival marker gene either fused to a coding sequence for adegron or under transcriptional control of an minimal promoter asdescribed in more detail herein. Further, the engine/editing vector mayalso and preferably does comprise a barcode. In some embodiments, theengine vector and editing vector may be separate. In this instance, thesurvival marker fused to a degron or under transcriptional control of aminimal promoter is on the editing vector comprising the gRNA and donorDNA. Further, the engine and editing vectors (whether separate orcombined) comprise control sequences operably linked to, e.g., thenuclease coding sequence, recombineering system coding sequences (ifpresent), donor nucleic acid, guide nucleic acid(s), and antibioticresistance gene(s).

Nuclease-Directed Genome Editing Generally

The automated instruments and methods described herein performnuclease-directed genome editing, introducing typically tens, tohundreds, to thousands, to tens of thousands of edits to a population ofyeast cells. In some embodiments, recursive cell editing is performedwhere edits are introduced in successive rounds of editing. A nucleicacid-guided nuclease or nuclease fusion complexed with an appropriatesynthetic guide nucleic acid in a cell can cut the genome of the cell ata desired location. The guide nucleic acid helps the nucleic acid-guidednuclease or nuclease fusion recognize and cut the DNA at a specifictarget sequence (either a cellular target sequence or a curing targetsequence). By manipulating the nucleotide sequence of the guide nucleicacid, the nucleic acid-guided nuclease or nuclease fusion may beprogrammed to target any DNA sequence for cleavage as long as anappropriate protospacer adjacent motif (PAM) is nearby. In certainaspects, the nucleic acid-guided nuclease editing system may use twoseparate guide nucleic acid molecules that combine to function as aguide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activatingCRISPR RNA (tracrRNA). In other aspects, the guide nucleic acid may be asingle guide nucleic acid that includes both the crRNA and tracrRNAsequences.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease or nuclease fusion and can thenhybridize with a target sequence, thereby directing the nuclease to thetarget sequence. A guide nucleic acid can be DNA or RNA; alternatively,a guide nucleic acid may comprise both DNA and RNA. In some embodiments,a guide nucleic acid may comprise modified or non-naturally occurringnucleotides. In cases where the guide nucleic acid comprises RNA, thegRNA may be encoded by a DNA sequence on a polynucleotide molecule suchas a plasmid, linear construct, or the coding sequence may andpreferably does reside within an editing cassette and is optionallyunder the control of an inducible promoter as described below. Foradditional information regarding “CREATE” editing cassettes, see U.S.Pat. Nos. 9,982,278; 10,266,849; 10,240,167; 10,351,877; 10,364,442;10,435,715; and 10,465,207 and U.S. Ser. No. 16/551,517; 16/773,618; and16/773,712, all of which are incorporated by reference herein.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease ornuclease fusion to the target sequence. The degree of complementaritybetween a guide sequence and the corresponding target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences. In some embodiments, a guide sequenceis about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or morenucleotides in length. In some embodiments, a guide sequence is lessthan about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length.Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15,16, 17, 18, 19, or 20 nucleotides in length.

In the present methods and compositions, the guide nucleic acids areprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript optionally under the control of an inducible promoter. Theguide nucleic acids are engineered to target a desired target sequence(either cellular target sequence or curing target sequence) by alteringthe guide sequence so that the guide sequence is complementary to adesired target sequence, thereby allowing hybridization between theguide sequence and the target sequence. In general, to generate an editin the target sequence, the gRNA/nuclease complex binds to a targetsequence as determined by the guide RNA, and the nuclease or nucleasefusion recognizes a protospacer adjacent motif (PAM) sequence adjacentto the target sequence. The target sequence can be any polynucleotideendogenous or exogenous to a yeast cell, or in vitro. For example, thetarget sequence can be a polynucleotide residing in the nucleus of anyeukaryotic cell. A target sequence can be a sequence encoding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., a regulatorypolynucleotide, an intron, a PAM, or “junk” DNA).

The guide nucleic acid may be and preferably is part of an editingcassette that encodes the donor nucleic acid that targets a cellulartarget sequence. Alternatively, the guide nucleic acid may not be partof the editing cassette and instead may be encoded on the editing vectorbackbone. For example, a sequence coding for a guide nucleic acid can beassembled or inserted into a vector backbone first, followed byinsertion of the donor nucleic acid in, e.g., an editing cassette. Inother cases, the donor nucleic acid in, e.g., an editing cassette can beinserted or assembled into a vector backbone first, followed byinsertion of the sequence coding for the guide nucleic acid. Preferably,the sequence encoding the guide nucleic acid and the donor nucleic acidare located together in a rationally-designed editing cassette and aresimultaneously inserted or assembled into a vector backbone to create anediting vector. In yet other embodiments, the sequence encoding theguide nucleic acid and the sequence encoding the donor nucleic acid areboth included in the editing cassette.

The target sequence is associated with a proto-spacer mutation (PAM),which is a short nucleotide sequence recognized by the gRNA/nucleasecomplex. The precise preferred PAM sequence and length requirements fordifferent nucleic acid-guided nucleases or nuclease fusions vary;however, PAMs typically are 2-7 base-pair sequences adjacent or inproximity to the target sequence and, depending on the nuclease, can be5′ or 3′ to the target sequence. Engineering of the PAM-interactingdomain of a nucleic acid-guided nuclease or nuclease fusion may allowfor alteration of PAM specificity, improve target site recognitionfidelity, decrease target site recognition fidelity, or increase theversatility of a nucleic acid-guided nuclease or nuclease fusion.

In certain embodiments, the genome editing of a cellular target sequenceboth introduces a desired DNA change to a cellular target sequence,e.g., the genomic DNA of a cell, and removes, mutates, or rendersinactive a proto-spacer mutation (PAM) region in the cellular targetsequence. Rendering the PAM at the cellular target sequence inactiveprecludes additional editing of the cell genome at that cellular targetsequence, e.g., upon subsequent exposure to a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid in later roundsof editing. Thus, cells having the desired cellular target sequence editand an altered PAM can be selected for by using a nucleic acid-guidednuclease or nuclease fusion complexed with a synthetic guide nucleicacid complementary to the cellular target sequence. Cells that did notundergo the first editing event will be cut rendering a double-strandedDNA break, and thus will not continue to be viable. The cells containingthe desired cellular target sequence edit and PAM alteration will not becut, as these edited cells no longer contain the necessary PAM site andwill continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases ornuclease fusions can recognize is constrained by the need for a specificPAM to be located near the desired target sequence. As a result, itoften can be difficult to target edits with the precision that isnecessary for genome editing. It has been found that nucleases andnuclease fusions can recognize some PAMs very well (e.g., canonicalPAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs).Because the methods disclosed herein allow for identification of editedcells in a background of unedited cells, the methods allow foridentification of edited cells where the PAM is less than optimal; thatis, the methods for identifying edited cells herein allow foridentification of edited cells even if editing efficiency is very low.Additionally, the present methods expand the scope of target sequencesthat may be edited since edits are more readily identified, includingcells where the genome edits are associated with less functional PAMs.

As for the nuclease or nuclease fusion component of the nucleicacid-guided nuclease editing system, a polynucleotide sequence encodingthe nucleic acid-guided nuclease or nuclease fusion can be codonoptimized for expression in particular cell types, such as yeast cells.The choice of nucleic acid-guided nuclease or nuclease fusion to beemployed depends on many factors, such as what type of edit is to bemade in the target sequence and whether an appropriate PAM is locatedclose to the desired target sequence. Nucleases of use in the methodsdescribed herein include but are not limited to Cas 9, Cas 12/CpfI,MAD2, or MAD7 or other MADzymes. Nuclease fusion enzymes typicallycomprise a CRISPR nucleic acid-guided nuclease engineered to cut one DNAstrand in the target DNA rather than making a double-stranded cut, andthe nuclease portion is fused to a reverse transcriptase. For moreinformation on nickases and nuclease fusion editing see U.S. Ser. Nos.16/740,418; 16/740,420 and 16/740,421, all filed 11 Jan. 2020. As withthe guide nucleic acid, the nuclease or nuclease fusion is encoded by aDNA sequence on a vector and may be under the control of an induciblepromoter. In some embodiments, the promoter may be separate from but thesame as the promoter controlling transcription of the guide nucleicacid; that is, a separate promoter drives the transcription of thenuclease or nuclease fusion and guide nucleic acid sequences but the twopromoters may be the same type of promoter. Alternatively, the promotercontrolling expression of the nuclease or nuclease fusion may bedifferent from the promoter controlling transcription of the guidenucleic acid; that is, e.g., the nuclease may be under the control of,e.g., the pCYC1 promoter, and the guide nucleic acid may be under thecontrol of the, e.g., SNR52 promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid comprising homology to the cellular target sequence.The donor nucleic acid is on the same vector and even in the sameediting cassette as the guide nucleic acid and preferably is (but notnecessarily is) under the control of the same promoter as the editinggRNA (that is, a single promoter driving the transcription of both theediting gRNA and the donor nucleic acid). The donor nucleic acid isdesigned to serve as a template for homologous recombination with acellular target sequence nicked or cleaved by the nucleic acid-guidednuclease as a part of the gRNA/nuclease complex. A donor nucleic acidpolynucleotide may be of any suitable length, such as about or more thanabout 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides in length.In certain preferred aspects, the donor nucleic acid can be provided asan oligonucleotide of between 20-300 nucleotides, more preferablybetween 50-250 nucleotides. The donor nucleic acid comprises a regionthat is complementary to a portion of the cellular target sequence(e.g., a homology arm). When optimally aligned, the donor nucleic acidoverlaps with (is complementary to) the cellular target sequence by,e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides.In many embodiments, the donor nucleic acid comprises two homology arms(regions complementary to the cellular target sequence) flanking themutation or difference between the donor nucleic acid and the cellulartarget sequence. The donor nucleic acid comprises at least one mutationor alteration compared to the cellular target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the cellular target sequence.

As noted supra, the donor nucleic acid is preferably provided as part ofa rationally-designed editing cassette or CREATE cassette, which isinserted into an editing vector backbone where the editing vectorbackbone may comprise a promoter driving transcription of the editinggRNA and the donor DNA. Moreover, there may be more than one, e.g., two,three, four, or more gRNA/donor nucleic acid pairs inserted into anediting vector (alternatively, a single rationally-designed editingcassette may comprise two to several editing gRNA/donor DNA pairs),where each editing gRNA is under the control of separate differentpromoters, separate like promoters, or where all gRNAs/donor nucleicacid pairs are under the control of a single promoter. In someembodiments the promoter driving transcription of the editing gRNA andthe donor nucleic acid (or driving more than one editing gRNA/donornucleic acid pair) is optionally an inducible promoter and the promoterdriving transcription of the nuclease optionally is an induciblepromoter as well. In some embodiments and preferably, the nuclease andediting gRNA/donor DNA are under the control of the same promoter.

Inducible editing is advantageous in that singulated cells can be grownfor several to many cell doublings before editing is initiated, whichincreases the likelihood that cells with edits will survive, as thedouble-strand cuts caused by active editing are largely toxic to thecells. This toxicity results both in cell death in the edited colonies,as well as possibly a lag in growth for the edited cells that do survivebut must repair and recover following editing. However, once the editedcells have a chance to recover, the size of the colonies of the editedcells will eventually catch up to the size of the colonies of uneditedcells.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a cellular targetsequence—one or more PAM sequence alterations that mutate, delete orrender inactive the PAM site in the cellular target sequence. The PAMsequence alteration in the cellular target sequence renders the PAM site“immune” to the nucleic acid-guided nuclease and protects the cellulartarget sequence from further editing in subsequent rounds of editing ifthe same nuclease is used.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding cellulartarget sequence. The barcode typically comprises four or morenucleotides. In some embodiments, the editing cassettes comprise acollection or library editing gRNAs and of donor nucleic acidsrepresenting, e.g., gene-wide or genome-wide libraries of editing gRNAsand donor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an editing vector or plasmid encodingcomponents of the nucleic acid-guided nuclease system further encodes anucleic acid-guided nuclease comprising one or more nuclear localizationsequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs. In some embodiments, the engineered nucleasecomprises NLSs at or near the amino-terminus, NLSs at or near thecarboxy-terminus, or a combination.

The editing vector comprises (or, if the engine and editing vector areseparate, both the engine and editing vectors comprise) controlsequences operably linked to the component sequences to be transcribed.As stated above, the promoters driving transcription of one or morecomponents of the nucleic acid-guided nuclease editing system optionallyare inducible.

Increasing Efficiency of Editing in Yeast

The present disclosure is drawn to increasing the efficiency of nucleicacid-guided nuclease editing in yeast. It has been found that twodifferent approaches increase editing efficiency in yeast by placingselective pressure on yeast cells to replicate a plasmid or vectorcomprising the nuclease, guide nucleic acid (gRNA), and donor DNAsequences. The first approach involves transforming yeast cells with anediting plasmid or vector comprising a coding sequence for a nuclease ornuclease fusion enzyme, a coding sequence for a guide RNA (gRNA)compatible with the nuclease or nuclease fusion enzyme, a codingsequence for a donor DNA, an origin of replication, and a codingsequence for a fusion protein comprising a coding sequence for a degronfused 5′ to a coding sequence for a survival marker (e.g., an antibioticresistance gene). This first approach results in destabilizing thesurvival marker protein leading to a decreased half-life for thesurvival marker protein. The second approach involves transforming yeastcells with an editing plasmid or vector comprising a coding sequence fora nuclease or nuclease fusion or nuclease fusion enzyme, a codingsequence for a guide RNA (gRNA) compatible with the nuclease enzyme, acoding sequence for a donor DNA, an origin of replication, and a minimalpromoter driving transcription of a coding sequence for a survivalmarker. This second approach decreases the level of transcription of thesurvival marker gene.

As described above, the term “survival marker” refers to a codingsequence for a gene product that allows a cell expressing the geneproduct to survive in selective medium. That is, cells expressing thesurvival marker gene product can grow and proliferate in a selectivemedium and cells that do not express the survival marker gene productcannot grow and proliferate in the selective medium. Survival markersinclude the G418 resistance gene (allowing cells expressing this gene tobe able to grow in medium containing G418), the blasticidin resistancegene (allowing cells expressing this gene to be able to grow in mediumcontaining blasticidin), the nourseothricin acetyl transferase gene(allowing cells expressing this gene to be able to grow in mediumcontaining nourseothricin) and the hygromycin resistance gene (allowingcells expressing this gene to be able to grow in medium containinghygromycin). The key to both approaches is that the yeast cellstransformed with the editing vector express the survival marker proteinallowing the yeast cells to grow in selective medium; however, in theseyeast cells the survival marker protein is expressed at a low level—dueto, in the first instance, a short protein half-life, and due to, in thesecond instance, a low level of transcription/translation of thesurvival marker protein—such that selective pressure is placed on theseyeast cells to increase replication of the editing plasmid or vectorthereby increasing the number of copies of the nuclease, gRNA, and donorDNA present.

In the first approach, graphically depicted in FIG. 1A, a codingsequence for a degron is fused to the 5′ terminus of a coding region fora survival marker, in this case an antibiotic resistance gene (“AbxR”).The top-most construct (i) depicts the antibiotic resistance gene (AbxR)under the control of a eukaryotic constitutive promoter. A constitutivepromoter driving transcription of AbxR results in high proteinexpression (high transcription of the antibiotic resistance gene andthus high expression of the antibiotic resistance protein) and theantibiotic resistance protein has a regular half-life. That is, survivalmarker proteins typically are stable enough to sustain levels of theprotein required for normal growth under selective conditions. Below thefirst construct is a second construct (ii) depicting the antibioticresistance gene (AbxR) fused at its 5′ terminus to a degron (“Ubi”),where the transcription of the degron-AbxR fusion is under the controlof a eukaryotic constitutive promoter (e.g., the same eukaryoticconstitutive promoter as the first construct (i)). Fusing the survivalmarker gene (AbxR) to a degron (“Ubi”) under constitutive expressionresults in high protein expression; however, the degron promotesdestabilization/degradation of the AbxR protein.

A degron is a portion of a protein that is important in regulation ofprotein degradation rates. Known degrons include short amino acidsequences, structural motifs, and exposed amino acids located anywherein the protein. Degrons are present in a variety of organisms, from theN-degrons first characterized in yeast, to the PEST sequence of mouseornithine decarboxylase. Although there are many types of differentdegrons—and a high degree of variability amongst them—degrons are allsimilar for their involvement in regulating the rate of proteindegradation. Mechanisms of degradation are often deemed “ubiquitindependent” or “ubiquitin independent.” Ubiquitin-dependent degrons areso named because they are implicated in the polyubiquitination processfor targeting a protein to the proteasome. In contrast, ubiquitinindependent degrons are not necessary for polyubiquitination. Ubiquitinis a small regulator protein consisting of 76 amino acids and is foundubiquitously (hence the name) in most tissues of eukaryotic organisms.

The addition of ubiquitin to a substrate protein (in this case, asurvival marker protein/antibiotic resistance protein) is calledubiquitination and thereby marks the substrate protein for degradation.Ubiquitin degron tags of use in the present methods and compositionsinclude Ubi-R, a 228 ubiquitin sequence with an Arginine (R) appended onthe 3′ end; Ubi-M, a 228 bp ubiquitin sequence with a Methionine (M)appended on the 3′ end; Ubi-Q, a 228 bp ubiquitin sequence with aGlutamine (Q) appended on the 3′ end; and Ubi-E, a 228 bp ubiquitinsequence with a Methionine (M) appended on the 3′ end. In addition tothe ubiquitin tags, are ubiquitin independent degrons such as the ZF1degron, a 36 amino acid motif recognized by the SOCS-box protein ZIF-1,which binds to the elongin C subunit of an ECS ubiquitin ligase complex;the C-terminal phosphodegrons (CTPD) from the C. elegans OMA-1 protein;and conditional degrons (e.g., inducible degrons) such as the Ts-degronand lt-degrons, which are induced by temperature shift and function whenadded to the N terminus of a coding protein; the auxin inducible degron(AID), which is a small protein tag that interacts with an F-Boxubiquitin ligase complex in the presence of a small molecule calledauxin; DD-based degrons, which are induced by Shield-1 ligand binding;LID, which is induced by Shield-1 ligand binding and functions only whenfused to the C-terminus of a protein; the PSD and B-LID degrons, whichare blue light inducible degrons; and TIPI, which is a TEV proteaseexpression induced degron. (See, e.g., Chen, et al., Yeast Research,12(5):598-607 (2012).)

In the present degron construct (ii) of FIG. 1A, a coding sequence for aubiquitin/N-degron tag (“Ubi”) is fused to the N-terminus of the codingsequence for the survival marker protein (e.g., the antibioticresistance gene “AbxR”) resulting in a Ubi-AbxR fusion protein. Theubiquitin/N-degron tag is cleaved off the fusion protein in vivo(depicted by the “Pac Man”-like element), generating a protein with anN-terminal amino acid other than methionine. Certain destabilizingN-terminal amino acid residues (primarily arginine) decrease thehalf-life of the protein carrying the N-degron tag. Here, the ubiquitintag is cleaved off, exposing an arginine residue (“R”) at the 5′terminus of the survival marker/antibiotic resistance protein. Whenarginine (“R”) is exposed at the N-terminus of the survival markerprotein, the survival marker protein is degraded. Degradation of thesurvival marker protein places selective pressure on the yeast cell inselective medium; thus, the yeast cell must replicate the editingplasmid or vector—that is, increase the plasmid copy number—in order tosurvive. The increase in plasmid copy number leads to a concurrentincrease in the copy numbers of the expressed nuclease, the transcribedgRNA, and the donor DNA.

FIG. 1B is a representation of (i) a standard constitutive promoterdriving an antibiotic resistance gene and (ii) a minimal constitutivepromoter driving an antibiotic resistance gene. As an alternative to theapproach of destabilizing the survival marker protein to influenceplasmid copy number shown in FIG. 1A, the approach depicted in FIG. 1Binfluences plasmid copy number via the expression level of the survivalmarker gene. It has been found that expression levels of a protein canbe down-regulated by using inducible promoters and truncated or minimal(also termed “core”) promoters. Minimal or core promoters typicallycomprise the minimal portion of a promoter required to properly initiatetranscription, including one or more of a transcription start site, abinding site for RNA polymerase, a general transcription factor bindingsite such as a TATA box or B recognition element, and a downstream corepromoter element (DPE). Often, the minimal promoter is located between−35 to +35 of the transcription start site. Use of a minimal promotertypically guarantees that there is always at least a low amount oftranscription of the target gene (in this case the survival markergene). Table 1 lists a number of exemplary minimal constitutivepromoters and Table 2 lists weak constitutive promoters. (See, e.g.,Natsuma and Kanemaki, Ann. Rev. Genetics, 51:83-102 (2017).)

TABLE 1 Minimal Promoters SEQ Promoter ID Name Sequence No. Ura3-dTAACCCAACTGCACAGAACAAAAACCTGCA 1 GGAAACGAAGATAAATC pHIS3 ATTGGCATTATCACATAATGAATTATACAT 2 (100 bp) TATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAAC GAAGGCAAAG pHIS3 CTTCGAAGAATATACTAAAAAATGAGCAGG 3 (50 bp) CAAGATAAACGAAGGCAAAG pHIS3AATGAGCAGGCAAGATAAACGAAGGCAAAG 4 (30 bp) pHIS3 CAAGATAAACGAAGGCAAAG 5(20 bp) pTRP1 TTCGGTCGAAAAAAGAAAAGGAGAGGGCCA 6 (100 bp)AGAGGGAGGGCATTGGTGACTATTGAGCAC GTGAGTATACGTGATTAAGCACACAAAGGC AGCTTGGAGTpTRP1 TATTGAGCACGTGAGTATACGTGATTAAGC 7 (50 bp) ACACAAAGGCAGCTTGGAGTpTRP1 GTGATTAAGCACACAAAGGCAGCTTGGAGT 8 (30 bp) pTRP1ACACAAAGGCAGCTTGGAGT 9 (20 bp) pLEU2 TTTTCCAATAGGTGGTTAGCAATCGTCTTA 10(100 bp) CTTTCTAACTTTTCTTACCTTTTACATTTC AGCAATATATATATATATATTTCAAGGATATACCATTCTA pLEU2 TTTACATTTCAGCAATATATATATATATAT 11 (50 bp)TTCAAGGATATACCATTCTA pLEU2 ATATATATATTTCAAGGATATACCATTCTA 12 (30 bp)pLEU2 TTCAAGGATATACCATTCTA 13 (20 bp) pURA3GGTATATATACGCATATGTGGTGTTGAAGA 14 (100 bp)AACATGAAATTGCCCAGTATTCTTAACCCA ACTGCACAGAACAAAAACCTGCAGGAAACG AAGATAAATCpURA3 TCTTAACCCAACTGCACAGAACAAAAACCT 15 (50 bp) GCAGGAAACGAAGATAAATCpURA3 ACAAAAACCTGCAGGAAACGAAGATAAATC 16 (30 bp) pURA3GCAGGAAACGAAGATAAATC 17 (20 bp) pTEF1 TCAAGTTTCAGTTTCATTTTTCTTGTTCTA 18(100 bp) TTACAACTTTTTTTACTTCTTGCTCATTAG AAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA pTEF1 TGCTCATTAGAAAGAAAGCATAGCAATCTA 19 (50 bp)ATCTAAGTTTTAATTACAAA pTEF1 TAGCAATCTAATCTAAGTTTTAATTACAAA 20 (30 bp)pTEF1 ATCTAAGTTTTAATTACAAA 21 (20 bp) pHXT7TAAAATAATAAAACATCAAGAACAAACAAG 22 (100 bp)CTCAACTTGTCTTTTCTAAGAACAAAGAAT AAACACAAAAACAAAAAGTTTTTTTAATTT TAATCAAAAApHXT7 AACAAAGAATAAACACAAAAACAAAAAGTT 23 (50 bp) TTTTTAATTTTAATCAAAAApHXT7 ACAAAAAGTTTTTTTAATTTTAATCAAAAA 24 (30 bp) pHXT7TTTTTAATTTTAATCAAAAA 25 (20 bp)

TABLE 2 Weak Constitutive Promoters SEQ Promoter  ID Name Sequence No.pSSA1 TCGACAAATTGTTACGTTGTGCTTTGATTTCTAAAGCG 26CTTCTTCACCTGCAGGTTCTGAGCCCTAAGAAAAAAAATTTCCTTGGTTGAAAATGGCGGAAAAAAAAAATTCAGAAAAAGAAATAAAGCACGTGTGCGCGGTGTGTGGATGATGGTTTCATCATTGTCAACGGCATTTTCGTTCTTGTGGATTGTTGTAAACTTTCCAGAACATTCTAGAAAGAAAGCACACGGAACGTTTAGAAGCTGTCATTTGCGTTTTTTCTCCAGATTTTAGTTGAGAAAGTAATTAAATTATTCTTCTTTTTCCAGAACGTTCCATCGGCGGCAAAAGGGAGAGAAAGAACCCAAAAAGAAGGGGGGCCATTTAGATTAGCTGATCGTTTCGAGGACTTCAAGGTTATATAAGGGGTGGATTGATGTATCTTCGAGAAGGGATTGAGTTGTAGTTTCGTTTCCCAATTCTTACTTAAGTTGTTTTATTTTCTCTATTTGTAAGATAAGCACATCAAAAGAAAAGTAATCAAGTATTACAAGAAACAAAAATTCAAGTAAATAACAGATAAT pPDA1GAAATTCAAAACTCTCCAGACAAAGCCTGCCATTTGGC 27CAAGCAAGCTTTTGACGACGCTATTGCTGAGTTGGACACTCTGTCTGAAGAATCATACAAAGATAGCACACTTATCATGCAACTGCTAAGGGACAATTTAACCTTATGGACTTCAGACATGTCCGAGTCCGGTCAAGCTGAAGACCAACAACAACAACAACAACATCAGCAACAGCAGCCACCTGCTGCCGCCGAAGGTGAAGCACCAAAGTAAGTATTCTGATAAATCTAAAGAGAAATTACTAAAAAAAAGAAAAAAAAAAGAACGGGGGTGTAATAATTTGTAGTTCATTATTGCAATTATATATCTATATCTATATATGTATATAACATTAACATGTGCATGTACACACGTAATCGCGCGTGTACATGTCTATATGTGTTACTTGAACTATACTGTTTTGACGTGTATGTTTATTTATCTCTCTTCTGATTCCTCCACCCCTTCCTTACTCAACCGGGTAAATGTCGCATCATGACTCCCGACAATAATCCCCTCTGGTATAGCGAGAAGCAACTTTAGCTTCTTAACGGCAAGAACTTTTTTATGTTTGTCGCACCTGTATCTTCACAAAAGTTGGATACAGCAATAAGAAAGGAAACCACAT TTGTGCCA pCYC1AGAAAGATGTCAACTGAAAAAAAAAAAGGTGAACACAG 28GAAAAAAAATAAAAAAAAAAAAAAAAAAAGGAGGACGAAACAAAAAAGTGAAAAAAAATGAAAATTTTTTTGGAAAACCAAGAAATGAATTATATTTCCGTGTGAGACGACATCGTCGAATATGATTCAGGGTAACAGTATTGATGTAATCAATTTCCTACCTGAATCTAAAATTCCCGGGAGCAAGATCAAGATGTTTTCACCGATCTTTCCGGTCTCTTTGGCCGGGGTTTACGGACGATGGCAGAAGACCAAAGCGCCAGTTCATTTGGCGAGCGTTGGTTGGTGGATCAAGCCCACGCGTAGGCAATCCTCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACAC ACAAACACAAATACACACACTAAATTAATApTPS1 AACCCGGTCTCGAAGAACATCAGCACCACGCCCGCAAC 29GACAAAGAACATTGCAATACACTTGCATATGTGAGCATAGTCGAGCGGTCCGTTCTGTGGTTGATGCTGTTGTTCTTTCTTCTGTTTGTCAGGGGTGATAGCCATATCTTCGTGCTCTTGTTGCGATTGTTCTGTTCCATCTGCACCAGAACAAAGAACAAAAGAACAAGGAACAAAGTCCAAGCACGTCAGCGCTGTTTATAAGGGGATTGACGAGGGATCGGGCCTAGAGTGCCAGCGCGCCAGGGAGAGGGAGCCCCCTGGGCCCTCATCCGCAGGCTGATAGGGGTCACCCCGCTGGGCAGGTCAGGGCAGGGGCTCTCAGGGGGGCGCCATGGACAAACTGCACTGAGGTTCTAAGACACATGTATTATTGTGAGTATGTATATATAGAGAGAGATTAAGGCGTACAGCCGGGTGGTAGAGATTGATTAACTTGGTAGTCTTATCTTGTCAATTGAGTTTCTGTCAGTTTCTTCTTGAACAAGCACGCAGCTAAGTAAGCAACAAAGCAGGCTAACAAACTAGGTAC TCACATACAGACTTATTAAGACATAGAACTpSSB1 CAGAGGAGTACACACGGGACTTGATCGAACAGATCGTG 30TTACAGTTGCGCTCGCAAAGAATGAAAATGGTTCAGACAAAGGATCAGTTCCTATTTATCTACCATGCTGCCAAGTATCTTAACAGTCTTTCCGTGAACCAATAGACAGCTATATAAAAGTTCCTAATTGTGCATTTTTTCAATAACAATACTTATTCATCCTTATAATTATATTCTAGCTTCGTTGTCATGGGAACATAGCCCATACACCGCAGTTATTTATGATCATTTCGAACGGGAAGTATGGATGAATCTTTTTTTTTTTTTTTTTATAGCACGCAACTGAAAAAAAAAAAAAGAAAAATTTTTCATCTTCGCTCGACGTTTCTTTTGTAGTACTCATCTCTTTTTATATAAAGATTAATTAGTTATTGTCGCTTTGCTTTTCCTTCTTTAAAAAATGTTTCTTGCTTTTGGATTTTCAGATGTCCCAAGATCATTACAGTATTTTAATTG AACAAA

As noted in FIG. 1B at (i), the survival marker/antibiotic resistancegene (“AbxR”) under the control of a standard constitutive promoterresults in high protein expression, normal protein half-life, and a lowplasmid copy number. Again, because the survival marker/antibioticresistance gene is transcribed at a high rate and thus the survivalmarker/antibiotic resistance protein is expressed at a high rate, thereis little to no selective pressure on the yeast cell harboring theediting vector or plasmid to increase replication of the editing vectoror plasmid, and thus there is no attendant increase in the expression ofthe nuclease, transcription of the gRNA, or transcription of the donorDNA.

However, in FIG. 1B at (ii), instead of a standard constitutivepromoter, a minimal promoter is used resulting in reduced transcriptionof the survival marker/antibiotic resistance gene (“AbxR”), and thusreduced expression of the survival marker/antibiotic resistance protein.Here, because the survival marker/antibiotic resistance gene istranscribed at a low rate and thus the survival marker/antibioticresistance protein is expressed at a low rate, there is increasedselective pressure on the yeast cell harboring the editing vector orplasmid to increase replication of the editing vector or plasmid.Increasing replication of the plasmid not only allows the yeast cell tosurvive, but additionally increases the expression of the nuclease,transcription of the gRNA, and copies of the donor DNA.

FIG. 1C is an exemplary vector map of a yeast 2-μ plasmid configured fornucleic acid-guided nuclease editing of a yeast genome, where anantibiotic resistance gene is fused at its 5′ end to a degron. Asdescribed in relation to FIG. 1A, such an editing plasmid or vectorresults in an increase in plasmid copy number in the host yeast cell.Beginning at 12 o'clock, there is a 2μ origin of replication; an SNR52promoter driving transcription of a gRNA and donor DNA (HA) with SNR52terminator element; a standard constitutive promoter drivingtranscription of a coding sequence for a degron-antibiotic resistancefusion gene with a terminator; a CYC1 promoter driving transcription ofa CRISPR enzyme or nuclease fusion coding sequence with a CYC1terminator; an ampicillin resistance gene to allow for selection inbacteria; and a pUC origin of replication for propagation of the editingvector in bacteria. As described above in relation to FIG. 1A, thefusion of the degron to the survival maker protein results indegradation of the survival marker protein, which in turn placesselective pressure on the yeast cell in selective medium; thus, theyeast cell must replicate the editing plasmid or vector—that is,increase the plasmid copy number—in order to survive. The increasedplasmid copy number leads to an increase in the copy numbers of theexpressed nuclease, the transcribed gRNA, and the donor DNA.

FIG. 1D is an exemplary vector map of a yeast 2-μ plasmid configured fornucleic acid-guided nuclease editing of a yeast genome, where theexpression of an antibiotic resistance gene is driven by a minimalconstitutive promoter. As described in relation to FIG. 1B and like thevector depicted in FIG. 1C, this editing plasmid or vector results in anincrease in plasmid copy number in the host yeast cell. Beginning at 12o'clock, there is a 2μ origin of replication; an SNR52 promoter drivingtranscription of a gRNA and donor DNA (HA) with SNR52 terminatorelement; a minimal promoter driving transcription of a coding sequencefor an antibiotic resistance gene with a terminator; a CYC1 promoterdriving transcription of a CRISPR enzyme or nuclease fusion codingsequence with a CYC1 terminator; an ampicillin resistance gene to allowfor selection in bacteria; and a pUC origin of replication forpropagation of the editing vector in bacteria. Again, because thesurvival marker/antibiotic resistance gene is transcribed at a low rateand thus the survival marker/antibiotic resistance protein is expressedat a low rate, there is increased selective pressure on the yeast cellharboring the editing vector or plasmid to increase replication of thevector or plasmid. Increasing replication of the plasmid not only allowsthe yeast cell to survive, but increases the expression of the nuclease,transcription of the gRNA, and copies of the donor DNA.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease Editing in Yeast Cells Automated Cell EditingInstruments

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument 200 to, e.g., perform one of the exemplary workflows fortargeted gene editing of live yeast cells. The instrument 200, forexample, may be and preferably is designed as a stand-alone desktopinstrument for use within a laboratory environment. The instrument 200may incorporate a mixture of reusable and disposable components forperforming the various integrated processes in conducting automatedgenome cleavage and/or editing in cells without human intervention.Illustrated is a gantry 202, providing an automated mechanical motionsystem (actuator) (not shown) that supplies XYZ axis motion control to,e.g., an automated (i.e., robotic) liquid handling system 258 including,e.g., an air displacement pipettor 232 which allows for cell processingamong multiple modules without human intervention. In some automatedmulti-module cell processing instruments, the air displacement pipettor232 is moved by gantry 202 and the various modules and reagentcartridges remain stationary; however, in other embodiments, the liquidhandling system 258 may stay stationary while the various modules andreagent cartridges are moved. Also included in the automatedmulti-module cell processing instrument 200 are reagent cartridges 210comprising reservoirs 212 and transformation module 230 (e.g., aflow-through electroporation device as described in detail in relationto FIGS. 5B-5F), as well as wash reservoirs 206, cell input reservoir251 and cell output reservoir 253. The wash reservoirs 206 may beconfigured to accommodate large tubes, for example, wash solutions, orsolutions that are used often throughout an iterative process. Althoughtwo of the reagent cartridges 210 comprise a wash reservoir 206 in FIG.2A, the wash reservoirs instead could be included in a wash cartridgewhere the reagent and wash cartridges are separate cartridges. In such acase, the reagent cartridge 210 and wash cartridge 204 may be identicalexcept for the consumables (reagents or other components containedwithin the various inserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. In someexamples, the robotic handling system 258 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips 215 may be provided in a pipettetransfer tip supply 214 for use with the air displacement pipettor 232.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises a cell growth vial 218 (described ingreater detail below in relation to FIGS. 3A-3D). Additionally seen isthe TFF module 222 (described above in detail in relation to FIGS.4A-4E). Also illustrated as part of the automated multi-module cellprocessing instrument 200 of FIG. 2A is a singulation module 240 (e.g.,a solid wall isolation, incubation and normalization device (SWIINdevice) is shown here) described herein in relation to FIGS. 6D-6G,served by, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Additionally seen is a selection module 220. Also note theplacement of three heatsinks 255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232. The deckof the multi-module cell processing instrument 200 may include aprotection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 200 are containedwithin a lip of the protection sink. Also seen are reagent cartridges210, which are shown disposed with thermal assemblies 211 which cancreate temperature zones appropriate for different regions. Note thatone of the reagent cartridges also comprises a flow-throughelectroporation device 230 (FTEP), served by FTEP interface (e.g.,manifold arm) and actuator 231. Also seen is TFF module 222 withadjacent thermal assembly 225, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 233. Thermal assemblies 225,235, and 245 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The rotating growth vial 218 iswithin a growth module 234, where the growth module is served by twothermal assemblies 235. Selection module is seen at 220. Also seen isthe SWIIN module 240, comprising a SWIIN cartridge 241, where the SWIINmodule also comprises a thermal assembly 245, illumination 243 (in thisembodiment, backlighting), evaporation and condensation control 249, andwhere the SWIIN module is served by SWIIN interface (e.g., manifold arm)and actuator 247. Also seen in this view is touch screen display 201,display actuator 203, illumination 205 (one on either side ofmulti-module cell processing instrument 200), and cameras 239 (oneillumination device on either side of multi-module cell processinginstrument 200). Finally, element 237 comprises electronics, such ascircuit control boards, high-voltage amplifiers, power supplies, andpower entry; as well as pneumatics, such as pumps, valves and sensors.

FIG. 2C illustrates a front perspective view of multi-module cellprocessing instrument 200 for use in as a desktop version of theautomated multi-module cell editing instrument 200. For example, achassis 290 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 290 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 290 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 2C, chassis 290 includestouch screen display 201, cooling grate 264, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 200 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis290 is lifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270a-270 c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, forexample, allow for additional air flow beneath the chassis 290.

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device, arotating growth vial 218 in a cell growth module 234, a tangential flowfiltration module 222, a SWIIN module 240 as well as interfaces andactuators for the various modules. In addition, chassis 290 housescontrol circuitry, liquid handling tubes, air pump controls, valves,sensors, thermal assemblies (e.g., heating and cooling units) and othercontrol mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242;10,421,959; 10,465,185; 10,519,437; 10,584,333; and 10,584,334 and U.S.Ser. No. 16/750,369, filed 23 Jan. 2020; Ser. No. 16/822,249, filed 18Mar. 2020; and Ser. No. 16/837,985, filed 1 Apr. 2020, all of which areherein incorporated by reference in their entirety.

The Rotating Cell Growth Module

FIG. 3A shows one embodiment of a rotating growth vial 300 for use withthe cell growth device and in the automated multi-module cell processinginstruments described herein. The rotating growth vial 300 is anoptically-transparent container having an open end 304 for receivingliquid media and cells, a central vial region 306 that defines theprimary container for growing cells, a tapered-to-constricted region 318defining at least one light path 310, a closed end 316, and a driveengagement mechanism 312. The rotating growth vial 300 has a centrallongitudinal axis 320 around which the vial rotates, and the light path310 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 310 is positioned in the lower constricted portion ofthe tapered-to-constricted region 318. Optionally, some embodiments ofthe rotating growth vial 300 have a second light path 308 in the taperedregion of the tapered-to-constricted region 318. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 310 is shorter than the second light path 308 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 308 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 312 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 312 such that the rotating growth vial 300 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 300 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 300 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 400 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 300 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 300 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 304with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 304 mayoptionally include an extended lip 302 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 300may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 300 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 300 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 300 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 300. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 300 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 3B is a perspective view of one embodiment of a cell growth device330. FIG. 3C depicts a cut-away view of the cell growth device 330 fromFIG. 3B. In both figures, the rotating growth vial 300 is seenpositioned inside a main housing 336 with the extended lip 302 of therotating growth vial 300 extending above the main housing 336.Additionally, end housings 352, a lower housing 332 and flanges 334 areindicated in both figures. Flanges 334 are used to attach the cellgrowth device 330 to heating/cooling means or other structure (notshown). FIG. 3C depicts additional detail. In FIG. 3C, upper bearing 342and lower bearing 340 are shown positioned within main housing 336.Upper bearing 342 and lower bearing 340 support the vertical load ofrotating growth vial 300. Lower housing 332 contains the drive motor338. The cell growth device 330 of FIG. 3C comprises two light paths: aprimary light path 344, and a secondary light path 350. Light path 344corresponds to light path 310 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 300, andlight path 350 corresponds to light path 308 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 316. Lightpaths 310 and 308 are not shown in FIG. 3C but may be seen in FIG. 3A.In addition to light paths 344 and 340, there is an emission board 348to illuminate the light path(s), and detector board 346 to detect thelight after the light travels through the cell culture liquid in therotating growth vial 300.

The motor 338 engages with drive mechanism 312 and is used to rotate therotating growth vial 300. In some embodiments, motor 338 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 338 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 336, end housings 352 and lower housing 332 of the cellgrowth device 330 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 300 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 330 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 330 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 330—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 330, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 3D illustrates a cell growth device 330 as part of an assemblycomprising the cell growth device 330 of FIG. 3B coupled to light source390, detector 392, and thermal components 394. The rotating growth vial300 is inserted into the cell growth device. Components of the lightsource 390 and detector 392 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 332 that houses the motor that rotatesthe rotating growth vial 300 is illustrated, as is one of the flanges334 that secures the cell growth device 330 to the assembly. Also, thethermal components 394 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 330 to the thermal components 394 via the flange 334 on the baseof the lower housing 332. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 300 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 300 by piercing though the foil seal orfilm. The programmed software of the cell growth device 330 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 300. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 300 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 330 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 330 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 330 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional details regarding rotating growth vials and cell growthdevices see U.S. Pat. Nos. 10,435,662; 10,443,031; 10,590,375 and U.S.Ser. No. 16/552,981, filed 7 Aug. 2019; Ser. No. 16/780,640, filed 3Feb. 2020.

The Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems for the processeslisted above is a module or component that can grow, perform bufferexchange, and/or concentrate cells and render them competent so thatthey may be transformed or transfected with the nucleic acids needed forengineering or editing the cell's genome.

FIG. 4A shows a retentate member 422 (top), permeate member 420 (middle)and a tangential flow assembly 410 (bottom) comprising the retentatemember 422, membrane 424 (not seen in FIG. 4A), and permeate member 420(also not seen). In FIG. 4A, retentate member 422 comprises a tangentialflow channel 402, which has a serpentine configuration that initiates atone lower corner of retentate member 422—specifically at retentate port428—traverses across and up then down and across retentate member 422,ending in the other lower corner of retentate member 422 at a secondretentate port 428. Also seen on retentate member 422 are energydirectors 491, which circumscribe the region where a membrane or filter(not seen in this FIG. 4A) is seated, as well as interdigitate betweenareas of channel 402. Energy directors 491 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 422 with permeate/filtrate member 420 via the energy directorcomponent 491 on permeate/filtrate member 420 (at right). Additionally,countersinks 423 can be seen, two on the bottom one at the top middle ofretentate member 422. Countersinks 423 are used to couple and tangentialflow assembly 410 to a reservoir assembly (not seen in this FIG. 4A butsee FIG. 4B).

Permeate/filtrate member 420 is seen in the middle of FIG. 4A andcomprises, in addition to energy director 491, through-holes forretentate ports 428 at each bottom corner (which mate with thethrough-holes for retentate ports 428 at the bottom corners of retentatemember 422), as well as a tangential flow channel 402 and twopermeate/filtrate ports 426 positioned at the top and center of permeatemember 420. The tangential flow channel 402 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 420 also comprisescountersinks 423, coincident with the countersinks 423 on retentatemember 420.

At bottom of FIG. 4A is a tangential flow assembly 410 comprising theretentate member 422 and permeate member 420 seen in this FIG. 4A. Inthis view, retentate member 422 is “on top” of the view, a membrane (notseen in this view of the assembly) would be adjacent and under retentatemember 422 and permeate member 420 (also not seen in this view of theassembly) is adjacent to and beneath the membrane. Again countersinks423 are seen, where the countersinks in the retentate member 422 and thepermeate member 420 are coincident and configured to mate with threadsor mating elements for the countersinks disposed on a reservoir assembly(not seen in FIG. 4A but see FIG. 4B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 μm. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 402 may vary depending on the volumeof the cell culture to be grown and the optical density of the cellculture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, orfrom 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400μm to 600 μm high. If the cross section of the flow channel 102 isgenerally round, oval or elliptical, the radius of the channel may befrom about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μmin hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, orfrom 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500μm in hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 420 members may be different depending on thedepth of the channel in each member.

FIG. 4B shows front perspective (top) and rear perspective (bottom)views of a reservoir assembly 450 configured to be used with thetangential flow assembly 410 seen in FIG. 4A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 450that is coupled to the tangential flow assembly 410 seen in FIG. 4A) areretentate reservoirs 452 on either side of permeate reservoir 454. Alsoseen are permeate ports 426, retentate ports 428, and three threads ormating elements 425 for countersinks 423 (countersinks 423 not seen inthis FIG. 4B). Threads or mating elements 425 for countersinks 423 areconfigured to mate or couple the tangential flow assembly 410 (seen inFIG. 4A) to reservoir assembly 450. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 410 to reservoir assembly 450. In additionis seen gasket 445 covering the top of reservoir assembly 450. Gasket445 is described in detail in relation to FIG. 4E. At left in FIG. 4B isa rear perspective view of reservoir assembly 450, where “rear” is theside of reservoir assembly 450 that is not coupled to the tangentialflow assembly. Seen are retentate reservoirs 452, permeate reservoir454, and gasket 445.

The TFF device may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 4C depicts a top-down view of the reservoir assemblies 450 shown inFIG. 4B. FIG. 4D depicts a cover 444 for reservoir assembly 450 shown inFIGS. 4B and 4E depicts a gasket 445 that in operation is disposed oncover 444 of reservoir assemblies 450 shown in FIG. 4B. FIG. 4C is atop-down view of reservoir assembly 450, showing the tops of the tworetentate reservoirs 452, one on either side of permeate reservoir 454.Also seen are grooves 432 that will mate with a pneumatic port (notshown), and fluid channels 434 that reside at the bottom of retentatereservoirs 452, which fluidically couple the retentate reservoirs 452with the retentate ports 428 (not shown), via the through-holes for theretentate ports in permeate member 420 and membrane 424 (also notshown). FIG. 4D depicts a cover 444 that is configured to be disposedupon the top of reservoir assembly 450. Cover 444 has round cut-outs atthe top of retentate reservoirs 452 and permeate/filtrate reservoir 454.Again at the bottom of retentate reservoirs 452 fluid channels 434 canbe seen, where fluid channels 434 fluidically couple retentatereservoirs 452 with the retentate ports 428 (not shown). Also shown arethree pneumatic ports 430 for each retentate reservoir 452 andpermeate/filtrate reservoir 454. FIG. 4E depicts a gasket 445 that isconfigured to be disposed upon the cover 444 of reservoir assembly 450.Seen are three fluid transfer ports 442 for each retentate reservoir 452and for permeate/filtrate reservoir 454. Again, three pneumatic ports430, for each retentate reservoir 452 and for permeate/filtratereservoir 454, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 406, collecting the cell culture through asecond retentate port 404 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 422)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 420) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 406.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall workflow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 420) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 404, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 406. All types of prokaryotic and eukaryoticcells—both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM,DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media maybe provided in a reagent cartridge as part of a kit.

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 404 while collecting the medium in one of thepermeate/filtrate ports 406 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeate ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 404 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 406 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 404 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 404 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 404 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port406 on the opposite end of the device/module from the permeate port 406that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 16/798,302, filed 22 Sep. 2020.

The Cell Transformation Module

FIGS. 5A and 5B depict the structure and components of an embodiment ofan exemplary reagent cartridge useful in the automated multi-moduleinstrument described therein. In FIG. 5A, reagent cartridge 500comprises a body 502, which has reservoirs 504. One reservoir 504 isshown empty, and two of the reservoirs have individual tubes (not shown)inserted therein, with individual tube covers 505. Additionally shownare rows of reservoirs into which have been inserted co-joined rows oflarge tubes 503 a, and co-joined rows of small tubes 503 b. Theco-joined rows of tubes are presented in a strip, with outer flanges 507that mate on the backside of the outer flange (not shown) with anindentation 509 in the body 502, so as to secure the co-joined rows oftubes (503 a and 503 b) to the reagent cartridge 500. Shown also is abase 511 of reagent cartridge body 502. Note that the reservoirs 504 inbody 502 are shaped generally like the tubes in the co-joined tubes thatare inserted into these reservoirs 504.

FIG. 5B depicts the reagent cartridge 500 in FIG. 5A with a row ofco-joined large tubes 503 a, a row of co-joined small tubes 503 b, andone large tube 560 with a cover 505 removed from (i.e., depicted above)the reservoirs 504 of the reagent cartridge 500. Again, the co-joinedrows of tubes are presented in a strip, with individual large tubes 561shown, and individual small tubes 555 shown. Again, each strip ofco-joined tubes comprises outer flanges 507 that mate on the backside(not shown) of the outer flange with an indentation 509 in the body 502,to secure the co-joined rows of tubes (503 a and 503 b) to the reagentcartridge 500. As in FIG. 5A, reagent cartridge body 502 comprises abase 511. Reagent cartridge 500 may be made from any suitable material,including stainless steel, aluminum, or plastics including polyvinylchloride, cyclic olefin copolymer (COC), polyethylene, polyamide,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),polysulfone, and polyurethane, and co-polymers of these and otherpolymers. Again, if reagent cartridge 500 is disposable, it preferablyis made of plastic. In addition, in many embodiments the material usedto fabricate the cartridge is thermally-conductive, as reagent cartridge500 may contact a thermal device (not shown) that heats or coolsreagents in the reagent reservoirs 504, including reagents in co-joinedtubes. In some embodiments, the thermal device is a Peltier device orthermoelectric cooler.

FIGS. 5C and 5D are top perspective and bottom perspective views,respectively, of an exemplary FTEP device 550 that may be part of (e.g.,a component in) reagent cartridge 500 in FIGS. 5A and 5B or may be astand-alone module; that is, not a part of a reagent cartridge or othermodule. FIG. 5C depicts an FTEP device 550. The FTEP device 550 haswells that define cell sample inlets 552 and cell sample outlets 554.FIG. 5D is a bottom perspective view of the FTEP device 550 of FIG. 5C.An inlet well 552 and an outlet well 554 can be seen in this view. Alsoseen in FIG. 5D are the bottom of an inlet 562 corresponding to well552, the bottom of an outlet 564 corresponding to the outlet well 554,the bottom of a defined flow channel 566 and the bottom of twoelectrodes 568 on either side of flow channel 566. The FTEP devices maycomprise push-pull pneumatic means to allow multi-pass electroporationprocedures; that is, cells to electroporated may be “pulled” from theinlet toward the outlet for one pass of electroporation, then be“pushed” from the outlet end of the FTEP device toward the inlet end topass between the electrodes again for another pass of electroporation.Further, this process may be repeated one to many times. For additionalinformation regarding FTEP devices, see, e.g., U.S. Pat. Nos.10,435,713; 10,443,074; 10,323,258; and 10,508,288. Further, otherembodiments of the reagent cartridge may provide or accommodateelectroporation devices that are not configured as FTEP devices, such asthose described in U.S. Ser. No. 16/109,156, filed 22 Aug. 2018. Forreagent cartridges useful in the present automated multi-module cellprocessing instruments, see, e.g., U.S. Pat. Nos. 10,376,889;10,406,525; 10,576,474; and U.S. Ser. No. 16/749,757, filed 22 Jan.2020; and Ser. No. 16/827,222, filed 23 Mar. 2020.

Additional details of the FTEP devices are illustrated in FIGS. 5E-5G.Note that in the FTEP devices in FIGS. 5E-5G the electrodes are placedsuch that a first electrode is placed between an inlet and a narrowedregion of the flow channel, and the second electrode is placed betweenthe narrowed region of the flow channel and an outlet. FIG. 5E shows atop planar view of an FTEP device 550 having an inlet 552 forintroducing a fluid containing cells and exogenous material into FTEPdevice 550 and an outlet 554 for removing the transformed cells from theFTEP following electroporation. The electrodes 568 are introducedthrough channels (not shown) in the device. FIG. 5F shows a cutaway viewfrom the top of the FTEP device 550, with the inlet 552, outlet 554, andelectrodes 568 positioned with respect to a flow channel 566. FIG. 5Gshows a side cutaway view of FTEP device 550 with the inlet 552 andinlet channel 572, and outlet 554 and outlet channel 574. The electrodes568 are positioned in electrode channels 576 so that they are in fluidcommunication with the flow channel 566, but not directly in the path ofthe cells traveling through the flow channel 566. Note that the firstelectrode is placed between the inlet and the narrowed region of theflow channel, and the second electrode is placed between the narrowedregion of the flow channel and the outlet. The electrodes 568 in thisaspect of the device are positioned in the electrode channels 576 whichare generally perpendicular to the flow channel 566 such that the fluidcontaining the cells and exogenous material flows from the inlet channel572 through the flow channel 566 to the outlet channel 574, and in theprocess fluid flows into the electrode channels 576 to be in contactwith the electrodes 568. In this aspect, the inlet channel, outletchannel and electrode channels all originate from the same planar sideof the device. In certain aspects, however, the electrodes may beintroduced from a different planar side of the FTEP device than theinlet and outlet channels.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit. Alternatively, the FTEP device may also be formed in two or moreparallel layers, e.g., a layer with the horizontal channel and filter, alayer with the vertical channels, and a layer with the inlet and outletports, which are manufactured and/or molded individually and assembledfollowing manufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 508 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired-as opposed to a disposable, one-use flow-through FTEP device-theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

As mentioned, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells to beelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,where the flow channel decreases in width, the flow channel may narrowto between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μmand 2 mm, or between 75 μm and 1 mm. The distance between the electrodesin the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall sizeof the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cmin length, or 4.5 cm to 10 cm in length. The overall width of the FTEPdevice may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cmto 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is wide enough so thatat least two cells can fit in the narrowed portion side-by-side. Forexample, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device. Flow-through electroporationdevices (either as a stand-alone instrument or as a module in anautomated multi-module system) are described in, e.g., U.S. Pat. Nos.10,435,713; 10,443,074; 10,323,258; and 10,508,288.

Cell Singulation and Enrichment Device

FIG. 6A depicts a solid wall device 6050 and a workflow for singulatingcells in microwells in the solid wall device. At the top left of thefigure (i), there is depicted solid wall device 6050 with microwells6052. A section 6054 of substrate 6050 is shown at (ii), also depictingmicrowells 6052. At (iii), a side cross-section of solid wall device6050 is shown, and microwells 6052 have been loaded, where, in thisembodiment, Poisson or substantial Poisson loading has taken place; thatis, each microwell has one or no cells, and the likelihood that any onemicrowell has more than one cell is low. At (iv), workflow 6040 isillustrated where substrate 6050 having microwells 6052 shows microwells6056 with one cell per microwell, microwells 6057 with no cells in themicrowells, and one microwell 6060 with two cells in the microwell. Instep 6051, the cells in the microwells are allowed to doubleapproximately 2-150 times to form clonal colonies (v), then editing isallowed to occur 6053.

After editing 6053, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 6058),where cells that do not undergo editing thrive (microwells 6059) (vi).All cells are allowed to continue grow to establish colonies andnormalize, where the colonies of edited cells in microwells 6058 catchup in size and/or cell number with the cells in microwells 6059 that donot undergo editing (vii). Once the cell colonies are normalized, eitherpooling 6060 of all cells in the microwells can take place, in whichcase the cells are enriched for edited cells by eliminating the biasfrom non-editing cells and fitness effects from editing; alternatively,colony growth in the microwells is monitored after editing, and slowgrowing colonies (e.g., the cells in microwells 6058) are identified andselected 6061 (e.g., “cherry picked”) resulting in even greaterenrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for yeast cell growth includes LB, SOC, TPD, YPG, YPAD,MEM and DMEM.

FIG. 6B is a photograph of one embodiment of a SWIIN. FIG. 6B is aphotograph of a SWIIN device with a permeable bottom on agar, on whichyeast cells have been singulated and grown into clonal colonies. FIG. 6Cpresents photographs of yeast colony growth in a SWIIN at various timepoints (at 0, 6, 11 and 32 hours).

A module useful for performing the methods depicted in FIG. 6A is asolid wall isolation, incubation, and normalization (SWIIN) module. FIG.6D depicts an embodiment of a SWIIN module 650 from an exploded topperspective view. In SWIIN module 650 the retentate member is formed onthe bottom of a top of a SWIIN module component and the permeate memberis formed on the top of the bottom of a SWIIN module component.

The SWIIN module 650 in FIG. 6D comprises from the top down, a reservoirgasket or cover 658, a retentate member 604 (where a retentate flowchannel cannot be seen in this FIG. 6D), a perforated member 601 swagedwith a filter (filter not seen in FIG. 6D), a permeate member 608comprising integrated reservoirs (permeate reservoirs 652 and retentatereservoirs 654), and two reservoir seals 662, which seal the bottom ofpermeate reservoirs 652 and retentate reservoirs 654. A permeate channel660 a can be seen disposed on the top of permeate member 608, defined bya raised portion 676 of serpentine channel 660 a, and ultrasonic tabs664 can be seen disposed on the top of permeate member 608 as well. Theperforations that form the wells on perforated member 601 are not seenin this FIG. 6D; however, through-holes 666 to accommodate theultrasonic tabs 664 are seen. In addition, supports 670 are disposed ateither end of SWIIN module 650 to support SWIIN module 650 and toelevate permeate member 608 and retentate member 604 above reservoirs652 and 654 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 660 a or the fluid path fromthe retentate reservoir to serpentine channel 660 b (neither fluid pathis seen in this FIG. 6D).

In this FIG. 6D, it can be seen that the serpentine channel 660 a thatis disposed on the top of permeate member 608 traverses permeate member608 for most of the length of permeate member 608 except for the portionof permeate member 608 that comprises permeate reservoirs 652 andretentate reservoirs 654 and for most of the width of permeate member608. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 660 a and 660 b can have approximately the samevolume or a different volume. For example, each “side” or portion 660 a,660 b of the serpentine channel may have a volume of, e.g., 2 mL, orserpentine channel 660 a of permeate member 608 may have a volume of 2mL, and the serpentine channel 660 b of retentate member 604 may have avolume of, e.g., 3 mL. The volume of fluid in the serpentine channel mayrange from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIINmodule comprising a, e.g., 50-500K perforation member). The volume ofthe reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, orfrom 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of allreservoirs may be the same or the volumes of the reservoirs may differ(e.g., the volume of the permeate reservoirs is greater than that of theretentate reservoirs).

The serpentine channel portions 660 a and 660 b of the permeate member608 and retentate member 604, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness.Embodiments the retentate (and permeate) members may be fabricated fromPMMA (poly(methyl methacrylate) or other materials may be used,including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 6G and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Automatedcolony pickers may be employed, such as those sold by, e.g., TECAN(Pickolo™ system, Mannedorf, Switzerland); Hudson Inc. (RapidPick™Springfield, N.J.); Molecular Devices (QPix 400™ system, San Jose,Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 650 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 650, or by applying atransparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g., FIG. 6G and the descriptionthereof infra.

In SWIIN module 650 cells and medium—at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel660 b from ports in retentate member 604, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 660 a in permeate member 608. The cells are retained in themicrowells of perforated member 601 as the cells cannot travel throughfilter 603. Appropriate medium may be introduced into permeate member608 through permeate ports 611. The medium flows upward through filter603 to nourish the cells in the microwells (perforations) of perforatedmember 601. Additionally, buffer exchange can be effected by cyclingmedium through the retentate and permeate members. In operation, thecells are deposited into the microwells, are grown for an initial, e.g.,2-100 doublings, editing may be induced by, e.g., raising thetemperature of the SWIIN to 42° C. to induce a temperature-induciblepromoter or by removing growth medium from the permeate member andreplacing the growth medium with a medium comprising a chemicalcomponent that induces an inducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module650 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 660 a and thus to filter 603 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 6E is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 6E, it canbe seen that serpentine channel 660 a is disposed on the top of permeatemember 608 is defined by raised portions 676 and traverses permeatemember 608 for most of the length and width of permeate member 608except for the portion of permeate member 608 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 652can be seen). Moving from left to right, reservoir gasket 658 isdisposed upon the integrated reservoir cover 678 (cover not seen in thisFIG. 6E) of retentate member 604. Gasket 658 comprises reservoir accessapertures 632 a, 632 b, 632 c, and 632 d, as well as pneumatic ports 633a, 633 b, 633 c and 633 d. Also at the far left end is support 670.Disposed under permeate reservoir 652 can be seen one of two reservoirseals 662. In addition to the retentate member being in cross section,the perforated member 601 and filter 603 (filter 603 is not seen in thisFIG. 6E) are in cross section. Note that there are a number ofultrasonic tabs 664 disposed at the right end of SWIIN module 650 and onraised portion 676 which defines the channel turns of serpentine channel660 a, including ultrasonic tabs 664 extending through through-holes 666of perforated member 601. There is also a support 670 at the end distalreservoirs 652, 654 of permeate member 608.

FIG. 6F is a side perspective view of an assembled SWIIIN module 650,including, from right to left, reservoir gasket 658 disposed uponintegrated reservoir cover 678 (not seen) of retentate member 604.Gasket 658 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 658 comprises reservoir access apertures 632 a, 632 b, 632 c, and632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d. Also atthe far-left end is support 670 of permeate member 608. In addition,permeate reservoir 652 can be seen, as well as one reservoir seal 662.At the far-right end is a second support 670.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 660.

FIG. 6G depicts the embodiment of the SWIIN module in FIGS. 6D-6Ffurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 698 comprises a SWIIN module 650seen lengthwise in cross section, where one permeate reservoir 652 isseen. Disposed immediately upon SWIIN module 650 is cover 694 anddisposed immediately below SWIIN module 650 is backlight 680, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 682, which is disposed over a heatsink 684. In thisFIG. 6G, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 686 and heat sink 688, as well as twothermoelectric coolers 692, and a controller 690 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells as well as strains of cells thatare, e.g., temperature sensitive, etc., and allows use oftemperature-sensitive promoters. Temperature control allows forprotocols to be adjusted to account for differences in transformationefficiency, cell growth and viability. For more details regarding solidwall isolation incubation and normalization devices see U.S. Pat. Nos.10,533,152; 10,550,363; 10,532,324; 10,625,212; and U.S. Ser. No.16/597,826, filed 19 Oct. 2019; Ser. No. 16/597,831, filed 9 Oct. 2019;Ser. No. 16/693,630, filed 25 Nov. 2019; and Ser. No. 16/686,066, filed15 Nov. 2019.

Use of the Automated Multi-Module Yeast Cell Processing Instrument

FIG. 7 illustrates an embodiment of a multi-module cell processinginstrument. This embodiment depicts an exemplary system that performsrecursive gene editing on a yeast cell population. The cell processinginstrument 700 may include a housing 726, a reservoir for storing cellsto be transformed or transfected 702, and a cell growth module(comprising, e.g., a rotating growth vial) 704. The cells to betransformed are transferred from a reservoir to the cell growth moduleto be cultured until the cells hit a target OD. Once the cells hit thetarget OD, the growth module may cool or freeze the cells for laterprocessing or transfer the cells to a cell concentration/filtrationmodule 706 where the cells are subjected to buffer exchange and renderedelectrocompetent, and the volume of the cells may be reducedsubstantially. Once the cells have been concentrated to an appropriatevolume, the cells are transferred to electroporation device 708. Inaddition to the reservoir for storing cells 702, the multi-module cellprocessing instrument includes a reservoir for storing the vectorpre-assembled with editing oligonucleotide cassettes 722. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 708, which already contains the cell culturegrown to a target OD. In the electroporation device 708, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into a recovery and dilution module 710, where thecells recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 712,where the cells can be stored at, e.g., 4° C. for later processing, orthe cells may be diluted and transferred to aselection/singulation/growth/induction/editing/normalization (SWIIN)module 720. In the SWIIN 720, the cells are arrayed such that there isan average of one cell per microwell. The arrayed cells may be inselection medium to select for cells that have been transformed ortransfected with the editing vector(s). Once singulated, in oneembodiment the cells grow through 2-50 doublings and establish colonies.Once colonies are established, editing is induced by providingconditions (e.g., temperature, addition of an inducing or repressingchemical) to induce editing. Editing is then initiated and allowed toproceed, the cells are allowed to grow to terminal size (e.g.,normalization of the colonies) in the microwells and then are treated toconditions that cure the editing vector from this round. In anotherembodiment editing is not induced and the cells are grown, allowed toedit, recover and normalize, and optionally cure.

Once cured, the cells can be flushed out of the microwells and pooled,then transferred to the storage (or recovery) unit 712 or can betransferred back to the growth module 704 for another round of editing.In between pooling and transfer to a growth module, there typically isone or more additional steps, such as cell recovery, medium exchange(rendering the cells electrocompetent), cell concentration (typicallyconcurrently with medium exchange by, e.g., filtration. Note that theselection/singulation/growth/induction/editing/normalization modules maybe the same module, where all processes are performed in, e.g., a solidwall device, or selection and/or dilution may take place in a separatevessel before the cells are transferred to the solid wallsingulation/growth/induction/editing/normalization/editing module(SWIIN). Similarly, the cells may be pooled after normalization,transferred to a separate vessel, and cured in the separate vessel. Asan alternative to singulation in, e.g., a solid wall device, thetransformed cells may be grown in—and editing can be induced in—bulkliquid as described in U.S. Ser. No. 16/399,988, filed 30 Apr. 2019.Once the putatively-edited cells are pooled, they may be subjected toanother round of editing, beginning with growth, cell concentration andtreatment to render electrocompetent, and transformation by yet anotherdonor nucleic acid in another editing cassette via the electroporationmodule 708.

In electroporation device 708, the yeast cells selected from the firstround of editing are transformed by a second set of editing oligos (orother type of oligos) and the cycle is repeated until the cells havebeen transformed and edited by a desired number of, e.g., editingcassettes. The multi-module cell processing instrument exemplified inFIG. 7 is controlled by a processor 724 configured to operate theinstrument based on user input or is controlled by one or more scriptsincluding at least one script associated with the reagent cartridge. Theprocessor 724 may control the timing, duration, and temperature ofvarious processes, the dispensing of reagents, and other operations ofthe various modules of the instrument 700. For example, a script or theprocessor may control the dispensing of cells, reagents, vectors, andediting oligonucleotides; which editing oligonucleotides are used forcell editing and in what order; the time, temperature and otherconditions used in the recovery and expression module, the wavelength atwhich OD is read in the cell growth module, the target OD to which thecells are grown, and the target time at which the cells will reach thetarget OD. In addition, the processor may be programmed to notify a user(e.g., via an application) as to the progress of the cells in theautomated multi-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 7, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.

Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined engine/editing vector) nonfunctional; diluting thevector(s) in the cell population via cell growth (that is, the moregrowth cycles the cells go through, the fewer daughter cells will retainthe editing or engine vector(s)), or by, e.g., utilizing aheat-sensitive origin of replication on the editing or engine vector (orcombined engine+editing vector). The conditions for curing will dependon the mechanism used for curing; that is, in this example, how thecuring plasmid cleaves the editing and/or engine vector.

FIG. 8 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising anisolation, induction, editing, and normalization module such as a SWIIN,or just by dilute plating on a solid substrate. The cell processinginstrument 800 may include a housing 826, a reservoir of cells to betransformed or transfected 802, and a growth module (a cell growthdevice) 804. The cells to be transformed are transferred from areservoir to the growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing, or the cells may betransferred to a cell concentration/filtration module 830 where thecells are rendered electrocompetent and concentrated to a volume optimalfor cell transformation. Once concentrated, the cells are thentransferred to an electroporation device 808 (e.g.,transformation/transfection module).

In addition to the reservoir for storing the cells, the system 800 mayinclude a reservoir for storing editing cassettes 816 and a reservoirfor storing an expression vector backbone 818. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module828, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 822 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 816 or 818. Once the processes carried out by thepurification module 822 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 808, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via concentration module 830. In electroporation device808, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 832. For examples of multi-module cell editinginstruments, see U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242;10,421,959; 10,465,185; 10,519,437; 10,584,333; and 10,584,334; and U.S.Ser. No. 16/750,369, filed 23 Jan. 2020; Ser. No. 16/822,249, filed 18Mar. 2020; and Ser. No. 16/837,985, filed 1 Apr. 2020, all of which areherein incorporated by reference in their entirety.

Following recovery, and, optionally, selection, the cells aretransferred to a growth, induction (optional), and editing module (bulkliquid culture) 840. The cells are allowed to grow until they go throughseveral to many doublings, then editing optionally is induced byinduction of transcription of one or both of the nuclease and gRNA. Insome embodiments, editing optionally is induced by transcription of oneor both of the nuclease and the gRNA being under the control of aninducible promoter. In some embodiments, the inducible promoter is a pLpromoter where the promoter is activated by a rise in temperature of thecell culture and “deactivated” by lowering the temperature of the cellculture.

The recovery, selection, isolation, growth, induction, editing andstorage modules may all be separate, may be arranged and combined asshown in FIG. 8, or may be arranged or combined in other configurations.In certain embodiments, recovery and selection are performed in onemodule, and isolation, growth, induction, editing, and re-growth areperformed in a separate module. Alternatively, recovery, selection,isolation, growth, induction, editing, and re-growth are performed in asingle module.

Once the cells are edited and re-grown (e.g., recovered from editing),the cells may be stored, e.g., in a storage module 812, where the cellscan be kept at, e.g., 4° C. until the cells are used in another round ofediting. The multi-module cell processing instrument is controlled by aprocessor 824 configured to operate the instrument based on user input,as directed by one or more scripts, or as a combination of user input ora script. The processor 824 may control the timing, duration,temperature, and operations of the various modules of the system 800 andthe dispensing of reagents. For example, the processor 824 may cool thecells post-transformation until editing is desired, upon which time thetemperature may be raised to a temperature conducive of genome editingand cell growth. The processor may be programmed with standard protocolparameters from which a user may select, a user may specify one or moreparameters manually or one or more scripts associated with the reagentcartridge may specify one or more operations and/or reaction parameters.In addition, the processor may notify the user (e.g., via an applicationto a smart phone or other device) that the cells have reached the targetOD as well as update the user as to the progress of the cells in thevarious modules in the multi-module system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was used togrow a yeast cell culture which was monitored in real time using anembodiment of the cell growth device described herein. The rotatinggrowth vial/cell growth device was used to measure OD₆₀₀ in real time ofyeast S. cerevisiae str. s288c cells in YPAD medium. The cells weregrown at 30° C. using oscillating rotation and employing a 2-paddlerotating growth vial. FIG. 9 is a graph showing the results. Note thatOD₆₀₀ 6.0 was reached in 14 hours.

Example II: Cell Concentration

The TFF module as described above in relation to FIGS. 4A-4E has beenused successfully to process and perform buffer exchange on yeastcultures. A yeast culture was initially concentrated to approximately 5ml using two passes through the TFF device in opposite directions. Thecells were washed with 50 ml of 1M sorbitol three times, with threepasses through the TFF device after each wash. After the third pass ofthe cells following the last wash with 1M sorbitol, the cells werepassed through the TFF device two times, wherein the yeast cell culturewas concentrated to approximately 525 μl. FIG. 10 presents the filterbuffer exchange performance for yeast cells determined by measuringfiltrate conductivity and filter processing time. Target conductivity(˜10 μS/cm) was achieved in approximately 23 minutes utilizing three 50ml 1M sorbitol washes and three passes through the TFF device for eachwash. The volume of the cells was reduced from 20 ml to 525 μl. Recoveryof approximately 90% of the cells has been achieved.

Example III: Production and Transformation of Electrocompetent S.Cerevisiae

For testing transformation of the FTEP device in yeast, electrocompetentS. Cerevisiae cells were created using the methods as generally setforth in Bergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013).Briefly, YFAP media was inoculated for overnight growth, with 3 mlinoculate to produce 100 ml of cells. Every 100 ml of culture processedresulted in approximately 1 ml of competent cells. Cells were incubatedat 30° C. in a shaking incubator until they reached an OD600 of1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD600 of 150+/−20 per ml.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 11. The device showed better transformation and survivalof electrocompetent S. cerevisiae at 2.5 kV voltages as compared to theNEPAGENE™ method. Input is total number of cells that were processed.

Example IV: Sinjulation of Yeast Colonies in a Solid Wall Device

Electrocompetent yeast cells were transformed with a cloned library, anisothermal assembled library, or a process control sgRNA plasmid(escapee surrogate). Electrocompetent Saccharomyces cerevisiae cellswere prepared as follows: The afternoon before transformation was tooccur, 10 mL of YPAD was inoculated with the selected Saccharomycescerevisiae strain. The culture was shaken at 250 RPM and 30° C.overnight. The next day, 100 mL of YPAD was added to a 250-mL baffledflask and inoculated with the overnight culture (around 2 mL ofovernight culture) until the OD600 reading reached 0.3+/−0.05. Theculture was placed in the 30° C. incubator shaking at 250 RPM andallowed to grow for 4-5 hours, with the OD checked every hour. When theculture reached an OD600 of approximately 1.5, 50 mL volumes were pouredinto two 50 mL conical vials, then centrifuged at 4300 RPM for 2 minutesat room temperature. The supernatant was removed from all 50 mL conicaltubes, while avoiding disturbing the cell pellet. 50 mL of a LithiumAcetate/Dithiothreitol solution was added to each conical tube and thepellet was gently resuspended. Both suspensions were transferred to a250 mL flask and placed in the shaker; then shaken at 30° C. and 200 RPMfor 30 minutes.

After incubation was complete, the suspension was transferred to two50-mL conical vials. The suspensions then were centrifuged at 4300 RPMfor 3 minutes, then the supernatant was discarded. Following the lithiumacetate/Dithiothreitol treatment step, cold liquids were used and thecells were kept on ice until electroporation. 50 mL of 1 M sorbitol wasadded and the pellet was resuspended, then centrifuged at 4300 RPM, 3minutes, 4° C., after which the supernatant was discarded. The 1Msorbitol wash was repeated twice for a total of three washes. 50 μL of 1M sorbitol was added to one pellet, cells were resuspended, thentransferred to the other tube to suspend the second pellet. The volumeof the cell suspension was measured and brought to 1 mL with cold 1 Msorbitol. At this point the cells were electrocompetent and could betransformed with a cloned library, an isothermal assembled library, orprocess control sgRNA plasmids.

In brief, a required number of 2-mm gap electroporation cuvettes wereprepared by labeling the cuvettes and then chilling on ice. Theappropriate plasmid—or DNA mixture—was added to each correspondingcuvette and placed back on ice. 100 uL of electrocompetent cells wastransferred to each labelled cuvette, and each sample was electroporatedusing appropriate electroporator conditions. 900 uL of room temperatureYPAD Sorbitol media was then added to each cuvette. The cell suspensionwas transferred to a 14 ml culture tube and then shaken at 30° C., 250RPM for 3 hours. After a 3 hr recovery, 9 ml of YPAD containing theappropriate antibiotic, e.g., G418 or Hygromycin B, was added. At thispoint the transformed cells were processed in parallel in the solid walldevice and the standard plating protocol, so as to compare“normalization” in the sold wall device with the standard benchtopprocess. Immediately before cells the cells were introduced to thepermeable-bottom solid wall device, the 0.45 μM filter forming thebottom of the microwells was treated with a 0.1% TWEEN solution toeffect proper spreading/distribution of the cells into the microwells ofthe solid wall device. The filters were placed into a Swinnex FilterHolder (47 mm, Millipore®, SX0004700) and 3 ml of a solution with 0.85%NaCl and 0.1% TWEEN was pulled through the solid wall device and filterthrough using a vacuum. Different TWEEN concentrations were evaluated,and it was determined that for a 47 mm diameter solid wall device with a0.45 μM filter forming the bottom of the microwells, a pre-treatment ofthe solid wall device+filter with 0.1% TWEEN was preferred (data notshown).

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 ml of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereshaken for 15 minutes, then were transferred to a sterile tube, e.g., a50 ml conical centrifuge tube. The OD600 of the cell suspension wasmeasured; at this point the cells can be processed in different waysdepending on the purpose of the study. For example, if an ADE2 stopcodon mutagenesis library is used, successfully-edited cells shouldresult in colonies with a red color phenotype when the resuspended cellsare spread onto YPD agar plates and allowed to grow for 4-7 days. Thisphenotypic difference allows for a quantification of percentage ofedited cells and the extent of normalization of edited and uneditedcells.

Example V: Growth and Editing of S. cerevisae Under Selective Pressure

To compare the editing rate of yeast cells transformed with the vectorsdepicted in FIGS. 1C and 1D, as well as a “standard” vector without adegron fusion or minimal promoter, 10 mL of YPAD medium was inoculatedwith the IY19 strain of S. cerevisiae. The culture was incubated at 30°C. while shaking at 250 rpm overnight. The following day, the overnightculture was diluted to an OD600=0.3 in 100 ml in fresh YPAD media. Thediluted culture was placed in the 30° C. incubator shaking at 250 rpmand grown for 4-5 hours. When the culture reached OD600 of ˜1.5, cellswere harvested by centrifuging at 4300 RPM for 3 minutes at roomtemperature.

The harvested cells were resuspended in 50 ml of 100 mM lithiumacetate+10 mM DTT solution and conditioned by shaking at 30° C./200 rpmfor 30 minutes. The cells were then harvested after conditioning bycentrifuging at 4300 RPM for 3 minutes at room temperature. Followingcentrifugation, the cells were washed 3× with 50 mL of ice-cold 1 Msorbitol; at the end of the final wash, cells were resuspended with 1 mlof ice cold 1M sorbitol. 1000 of the cell resuspension was used in everytransformation (standard plasmid backbone, degron plasmid backbone (seeFIG. 1C) and minimal promoter plasmid backbone (see FIG. 1D)). Theediting cassettes were the same for each plasmid backbone. The competentcells were electroporated in a 2-mm electroporation cuvette along withthe appropriate editing plasmid.

The plasmid backbone/cassette mix comprised 500 ng of linear plasmidbackbone (standard, degron, and minimal promoter) and 50 ng of cassette.A Nepagene electroporator used the following conditions to electroporateeach sample: for the poring pulse: a single pulse at voltage=1800, pulselength=5.0, pulse interval=50.0 msec; for the transfer pulses, threepulses at voltage=100, pulse length=50.0 msec, pulse interval−50.0.Following electroporation, 900 μL of room temperature YPAD+1M Sorbitolmedia was added to each cuvette and the cell suspension was transferredto a pop-cap 15 mL tube. This mix was shaken at 30° C./250 RPM for 3hours. 1-10 μl of the transformation was then plated on YPD+G418 (200μg/ml) plates and incubated at 30° C. for 3 days. 96 colonies werepicked from transformation plates, and these colonies were cultured in 1mL of YPAD+G418 (200 μg/ml) media in a deep-well plate and grownovernight at 30° C. The 96-well plate was used for DNA extraction usinga SV lysis Promega DNA extraction kit (Promega, Madison, Wis.) accordingto manufacturer's instructions.

Three different survival marker proteins were tested: hygromycin (FIGS.12A, 12B and 12C), kanamycin (FIGS. 13A, 13B, and 13C), and blasticidin(data not shown). The minimal promoter was the truncated version of theURA3 promoter, URA3-d, comprising only 47 nucleotides located upstreamof the start codon. Using hygromycin as the survival marker, thestandard editing plasmid resulted in clonality of 1.4%, the plasmidcoding for the degron-hygromycin fusion protein resulted in clonality of10.2%, and the plasmid with the minimal promoter driving transcriptionof the hygromycin gene resulted in 32.6% clonality. Using kanamycin asthe survival marker, the standard editing plasmid resulted in clonalityof 14.7%, the plasmid coding for the degron-kanamycin fusion proteinresulted in clonality of 21.4%, and the plasmid with the minimalpromoter driving transcription of the kanamycin gene resulted in 39.3%clonality. Preliminary data with the minimal promoter drivingtranscription of blasticidin as the survival marker, the clonality was6.9% with 19/20 unique edits (data not shown).

Example VI: Growth and Editing of S. cerevisae Under Selective Pressure

FIG. 14 shows the plasmid copy number and edit rate of various plasmidconstructs and their edit rates measured via shotgun NGS sequencing. Theplasmid copy number is calculated by taking the ratio of median plasmidread depth vs. median genomic read depth. The histograms show themeasured plasmid copy number from at least 48 isolated colonies. Theedit rates were calculated via next gen sequencing of isolated coloniesand are the same values that appear in FIGS. 12A-12C and FIGS. 13A-13C.Note the correlation between plasmid copy number and observed edit rate,which illustrates the utility of increasing the plasmid copy number ofediting constructs to increase observed editing.

Example VII: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. An ampR plasmid backbone and a lacZ_F172* editing cassettewere assembled via Gibson Assembly® into an “editing vector” in anisothermal nucleic acid assembly module included in the automatedinstrument. lacZ_F172 functionally knocks out the lacZ gene.“lacZ_F172*” indicates that the edit happens at the 172nd residue in thelacZ amino acid sequence. Following assembly, the product was de-saltedin the isothermal nucleic acid assembly module using AMPure beads,washed with 80% ethanol, and eluted in buffer. The assembled editingvector and recombineering-ready, electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module), and allowed to recover inSOC medium containing chloramphenicol. Carbenicillin was added to themedium after 1 hour, and the cells were allowed to recover for another 2hours. After recovery, the cells were held at 4° C. until recovered bythe user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example VIII: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in anisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, 916.

We claim:
 1. An editing vector for performing nucleic acid-guidednuclease editing in yeast comprising: a yeast 2-μ backbone, a 2μ originof replication; a standard constitutive (e.g., non-minimal or non-core)promoter driving transcription of a gRNA sequence and donor DNA (HA)sequence followed by a terminator element 3′ to the gRNA and donor DNAsequences; a standard constitutive (e.g., non-minimal or non-core)promoter driving transcription of a coding sequence for adegron-survival marker fusion gene followed by a terminator element 3′to the degron-survival marker fusion gene; a standard constitutive(e.g., non-minimal or non-core) promoter driving transcription of anuclease coding sequence with a terminator element 5′ to the nucleasecoding sequence; and an origin of replication for propagation of theediting vector in bacteria.
 2. The editing vector of claim 1, whereinthe degron is an ubiquitin-dependent degron.
 3. The editing vector ofclaim 1, wherein the degron is selected from Ura3-d degon, Ubi-R degron,Ubi-M degron, Ubi-Q degron, Ubi-E degron, ZF1 degron, C-terminalphosphodegron; Ts-degron; lt-degron; auxin inducible degron; DD-degron,LID-degron; PSD degron, B-LID degron; or a TIPI degron.
 4. The editingvector of claim 2, wherein the degron is the Ura3-d degron.
 5. Theediting vector of claim 1, wherein the survival marker is selected fromthe group of hygromycin, blasticidin, kanamycin, or nourseothricin. 6.An editing vector for performing nucleic acid-guided nuclease editing inyeast comprising: a yeast 2-μ backbone, a 2μ origin of replication; astandard constitutive (e.g., non-minimal or non-core) promoter drivingtranscription of a gRNA sequence and donor DNA (HA) sequence withfollowed by a terminator element 3′ to the gRNA and donor DNA sequences;a minimal promoter driving transcription of a coding sequence for asurvival marker gene followed by a terminator element 3′ to the survivalmarker gene; a standard constitutive (e.g., non-minimal or non-core)promoter driving transcription of a nuclease coding sequence with aterminator element 5′ to the nuclease coding sequence; and an origin ofreplication for propagation of the editing vector in bacteria.
 7. Theediting vector of claim 5, wherein the minimal promoter is the URA3-dpromoter.
 8. The editing vector of claim 5, wherein the survival markeris selected from the group of hygromycin, blasticidin, kanamycin, ornourseothricin.